Studies of chemical carcinogenesis would be greatly facilitated by the availability of a chemically defined medium for growth in vitro of a normal cell type that frequently becomes malignant in vivo. The primary objectives of the proposed research are to define completely the growth requirements of normal human mammary epithelial cells and to develop a practical chemically-defined medium for their multiplication in culture. This will be accomplished by qualitative and quantitative optimization of the basal nutrient medium to reduce the requirement for undefined supplements, and by replacing the remaining requirements with a mixture of defined hormones, growth factors, carrier proteins, lipids, and any other growth-promoting substances that are found to be needed. The feasibility of this approach has already been demonstrated by development of a chemically defined medium for clonal growth of human epidermal keratinocytes. In addition to its value to research on carcinogenesis, a defined medium for normal human mammary epithelial cells would also be of major significance in studies of hormonal control over growth and functional differentiation of these cells in culture.